In Vivo Epigenomic Profiling of Germ Cells Reveals Germ Cell Molecular Signatures

1. Download : Download high-res image (85KB)
2. Download : Download full-size image

Highlights? Modified small-scale ChIP-seq method applicable to small number of cells ? Genome-wide maps of H3K4me3, H3K27me3, H3K27ac, and H2BK20ac of germ cells in vivo ? Identification of active and inactive regulatory elements in germ cells in vivo ? Germ cell H3K27me3 regions are enriched for retrotransposon repeats     

Ghrelin Receptor (GHS-R1A) Antagonism Suppresses Both Alcohol Consumption and the Alcohol Deprivation Effect in Rats following Long-Term Voluntary Alcohol Consumption

Alcohol dependence is a heterogeneous disorder where several signalling systems play important roles. Recent studies implicate that the gut-brain hormone ghrelin, an orexigenic peptide, is a potential mediator of alcohol related behaviours. Ghrelin increases whereas a ghrelin receptor (GHS-R1A) antagonist decreases alcohol consumption as well as operant self-administration of alcohol in rodents that have consumed alcohol for twelve weeks. In the present study we aimed at investigating the effect of acute and repeated treatment with the GHS-R1A antagonist JMV2959 on alcohol intake in a group of rats following voluntarily alcohol consumption for two, five and eight months. After approximately ten months of voluntary alcohol consumption the expression of the GHS-R1A gene (Ghsr) as well as the degree of methylation of a CpG island found in Ghsr was examined in reward related brain areas. In a separate group of rats, we examined the effect of the JMV2959 on alcohol relapse using the alcohol deprivation paradigm. Acute JMV2959 treatment was found to decrease alcohol intake and the effect was more pronounced after five, compared to two months of alcohol exposure. In addition, repeated JMV2959 treatment decreased alcohol intake without inducing tolerance or rebound increase in alcohol intake after the treatment. The GHS-R1A antagonist prevented the alcohol deprivation effect in rats. There was a significant down-regulation of the Ghsr expression in the ventral tegmental area (VTA) in high- compared to low-alcohol consuming rats after approximately ten months of voluntary alcohol consumption. Further analysis revealed a negative correlation between Ghsr expression in the VTA and alcohol intake. No differences in methylation degree were found between high- compared to low-alcohol consuming rats. These findings support previous studies showing that the ghrelin signalling system may constitute a potential target for development of novel treatment strategies for alcohol dependence.     

Trypsin inhibitor activity in mature tobacco and tomato plants is mainly induced locally in response to insect attack,wounding and virus infection

Wounding of plants by insects is often mimicked in the laboratory by mechanical means such as cutting or crushing, and has not been compared directly with other forms of biotic stress such as virus infection. To compare the response of plants to these types of biotic and abiotic stress, trypsin inhibitor (TI) activity induced locally and systemically in mature tobacco (Nicotiana tabacum L.) and tomato (Lycopersicon esculentum L.) plants was followed for 12 days. In tobacco, cutting, crushing and insect feeding all induced comparable levels of TI activity of approx. 5 nmol·(mg leaf protein)^?1 in wounded leaves, while tobacco mosaic virus (TMV) infection of tobacco induced 10-fold lower amounts in the infected leaves. In tomato, feeding by insects also led to the induction of a level of TI activity of 5 nmol·(mg leaf protein)^?1. In contrast, both cutting and crushing of tomato leaves induced 10-fold higher amounts. These data show that biotic stress, in the form of insect feeding and TMV infection, and abiotic stress, in the form of wounding, have different effects on local levels of induced TI activity in mature tobacco and tomato plants. Irrespective of the type of wounding, in neither tobacco nor tomato could systemic induction of TI activity be observed in nearby unwounded leaves, which suggests that systemic induction of TI activity in mature tobacco and tomato plants is different from systemic TI induction in seedlings. Wounding of tobacco leaves, however, did increase the responsiveness to wounding elsewhere in the plant, as measured by an increased induction of TI activity.     

Plant sphingolipids: structural diversity,biosynthesis, first genes and functions

In mammals and Saccharomyces cerevisiae, sphingolipids have been a subject of intensive research triggered by the interest in their structural diversity and in mammalian pathophysiology as well as in the availability of yeast mutants and suppressor strains. More recently, sphingolipids have attracted additional interest, because they are emerging as an important class of messenger molecules linked to many different cellular functions. In plants, sphingolipids show structural features differing from those found in animals and fungi, and much less is known about their biosynthesis and function. This review focuses on the sphingolipid modifications found in plants and on recent advances in the functional characterization of genes gaining new insight into plant sphingolipid biosynthesis. Recent studies indicate that plant sphingolipids may be also involved in signal transduction, membrane stability, host-pathogen interactions and stress responses.     

Testosterone and its effects on courtship in golden-collared manakins (Manacus vitellinus): seasonal, sex, and age differences

Male golden-collared manakins gather on leks and perform an acrobatic display to attract females. In temperate breeding species, testosterone (T) activation of courtship displays has been well studied. Few studies have examined T activation of displays in tropical species; even fewer have explored the activational role of T in elaborate courtship displays such as in the manakin. In some tropical species, including manakins, territorial aggression or song behavior are uncoupled from T. We have previously shown that T activates display behavior in manakin males when endogenous T levels are low in the non-courtship season. To understand how T functions in breeding birds, we examined T levels in a large group of manakins sampled during the courtship and non-courtship season. In addition, during the courtship season, we gave T implants to adult males, juvenile males, and females. We found that T levels were low during the non-courtship season and comparatively higher on average during the courtship season. However, T levels were low in many adult males during the courtship season, especially when compared to temperate breeding species. Regardless of initial endogenous T levels during the courtship season, T implants did not increase the display frequency of adult males. T-treated females and juvenile males did display under similar conditions. Our data suggest that the effects of T on manakin display vary with season, sex, and age and that high T is not necessary for display.     

Conformational gating of dimannose binding to the antiviral protein cyanovirin revealed from the crystal structure at 1.35 A resolution

Cyanovirin (CV-N) is a small lectin with potent HIV neutralization activity, which could be exploited for a mucosal defense against HIV infection. The wild-type (wt) protein binds with high affinity to mannose-rich oligosaccharides on the surface of gp120 through two quasi-symmetric sites, located in domains A and B. We recently reported on a mutant of CV-N that contained a single functional mannose-binding site, domain B, showing that multivalent binding to oligomannosides is necessary for antiviral activity. The structure of the complex with dimannose determined at 1.8 A resolution revealed a different conformation of the binding site than previously observed in the NMR structure of wt CV-N. Here, we present the 1.35 A resolution structure of the complex, which traps three different binding conformations of the site and provides experimental support for a locking and gating mechanism in the nanoscale time regime observed by molecular dynamics simulations.     

Prognosis of sentinel node staged patients with primary cutaneous melanoma

Background

This study investigated survival probabilities and prognostic factors in sentinel lymph node biopsy (SLNB) staged patients with cutaneous melanoma (CM) with the aim of defining subgroups of patients who are at higher risk for recurrences and who should be considered for adjuvant clinical trials.

Methods

Patients with primary CM who underwent SLNB in the Department of Dermatology, University of Tuebingen, Germany, between 1996 and 2009 were included into this study. Survival probabilities and prognostic factors were evaluated by Kaplan-Meier and multivariate Cox proportional hazard models.

Results

1909 SLNB staged patients were evaluated. Median follow-up time was 44 months. Median tumor thickness was 1.8 mm, ulceration was present in 31.8% of cases. The 5-year Overall Survival (OS) was 90.3% in SLNB negative patients (IB 96.2%, IIA 87.0%, IIB 78.1%, IIC 72.6%). Patients with micrometastases (stage IIIA/B) had a 5-year OS rate of 70.9% which was clearly less favorable than for stages I–II. Multivariate analysis revealed tumor thickness, ulceration, body site, histopathologic subtype and SLNB status as independent significant prognostic factors.

Conclusion

Survival rates of patients with primary CM in stages I–II were shown to be much more favorable than previously reported from non sentinel node staged collectives. For future clinical trials, sample size calculations should be adapted using survival probabilities based on sentinel node staging.     

Immunodominant Francisella tularensis antigens identified using proteome microarray

Stimulation of protective immune responses against intracellular pathogens is difficult to achieve using non-replicating vaccines. BALB/c mice immunized by intramuscular injection with killed Francisella tularensis (live vaccine strain) adjuvanted with preformed immune stimulating complexes admixed with CpG, were protected when systemically challenged with a highly virulent strain of F. tularensis (Schu S4). Serum from immunized mice was used to probe a whole proteome microarray in order to identify immunodominant antigens. Eleven out of the top 12 immunodominant antigens have been previously described as immunoreactive in F. tularensis. However, 31 previously unreported immunoreactive antigens were revealed using this approach. Twenty four (50%) of the ORFs on the immunodominant hit list belonged to the category of surface or membrane associated proteins compared to only 22% of the entire proteome. There were eight hypothetical protein hits and eight hits from proteins associated with different aspects of metabolism. The chip also allowed us to readily determine the IgG subclass bias, towards individual or multiple antigens, in protected and unprotected animals. These data give insight into the protective immune response and have potentially important implications for the rational design of non-living vaccines for tularemia and other intracellular pathogens.     

The Diversity of Yellow-Related Proteins in Sand Flies (Diptera: Psychodidae)

Yellow-related proteins (YRPs) present in sand fly saliva act as affinity binders of bioamines, and help the fly to complete a bloodmeal by scavenging the physiological signals of damaged cells. They are also the main antigens in sand fly saliva and their recombinant form is used as a marker of host exposure to sand flies. Moreover, several salivary proteins and plasmids coding these proteins induce strong immune response in hosts bitten by sand flies and are being used to design protecting vaccines against Leishmania parasites. In this study, thirty two 3D models of different yellow-related proteins from thirteen sand fly species of two genera were constructed based on the known protein structure from Lutzomyia longipalpis. We also studied evolutionary relationships among species based on protein sequences as well as sequence and structural variability of their ligand-binding site. All of these 33 sand fly YRPs shared a similar structure, including a unique tunnel that connects the ligand-binding site with the solvent by two independent paths. However, intraspecific modifications found among these proteins affects the charges of the entrances to the tunnel, the length of the tunnel and its hydrophobicity. We suggest that these structural and sequential differences influence the ligand-binding abilities of these proteins and provide sand flies with a greater number of YRP paralogs with more nuanced answers to bioamines. All these characteristics allow us to better evaluate these proteins with respect to their potential use as part of anti-Leishmania vaccines or as an antigen to measure host exposure to sand flies.     

Why boys will be boys: two pathways of fetal testicular androgen biosynthesis are needed for male sexual differentiation

Human sexual determination is initiated by a cascade of genes that lead to the development of the fetal gonad. Whereas development of the female external genitalia does not require fetal ovarian hormones, male genital development requires the action of testicular testosterone and its more potent derivative dihydrotestosterone (DHT). The "classic" biosynthetic pathway from cholesterol to testosterone in the testis and the subsequent conversion of testosterone to DHT in genital skin is well established. Recently, an alternative pathway leading to DHT has been described in marsupials, but its potential importance to human development is unclear. AKR1C2 is an enzyme that participates in the alternative but not the classic pathway. Using a candidate gene approach, we identified AKR1C2 mutations with sex-limited recessive inheritance in four 46,XY individuals with disordered sexual development (DSD). Analysis of the inheritance of microsatellite markers excluded other candidate loci. Affected individuals had moderate to severe undervirilization at birth; when recreated by site-directed mutagenesis and expressed in bacteria, the mutant AKR1C2 had diminished but not absent catalytic activities. The 46,XY DSD individuals also carry a mutation causing aberrant splicing in AKR1C4, which encodes an enzyme with similar activity. This suggests a mode of inheritance where the severity of the developmental defect depends on the number of mutations in the two genes. An unrelated 46,XY DSD patient carried AKR1C2 mutations on both alleles, confirming the essential role of AKR1C2 and corroborating the hypothesis that both the classic and alternative pathways of testicular androgen biosynthesis are needed for normal human male sexual differentiation.